Ceramide is a naturally occurring sphingolipid that has emerged as an important second messenger molecule in apoptosis signaling. The production of ceramide is so common during apoptosis induced by diverse stress stimuli that it has been considered a universal feature of this process. Ceramide is a potent apoptotic agent and important effector molecule of both cellular stress- and mitogenactivated signal transduction pathways. Ceramide is also a potent activator of a protein phosphatase that appears to be a member of the PP2A family. We have recently discovered that ceramide activates a mitochondrial PP2A and induces Bc12 dephosphorylation. We have also determined that there is a strong correlation between ceramide-induced Bc12 dephosphorylation and the promotion of cell death. Furthermore, we have now discovered that the non-phosphorylatable (i.e. PP2A-resistant), gain-of-function S70-E mutant Bc12 (which appears to mimic phosphorylation) can protect cells from ceramide-induced apoptosis at concentrations of ceramide (i.e.> 10 uM) where WT Bc12 is found to be dephosphorylated and fails to protect cells. These preliminary results suggest a novel, experimentally testable hypothesis by which ceramide may regulate Bc12 function and thereby affect the cell?s apoptotic potential during stress. The relative chemosensistivity of pre-B REH cells, which paradoxically display robust expression of Bc12, appears to be explained by overexpression of mitochondrial PP2A. Since the PP2A inhibitor, okadaic acid, promotes chemoresistance and Bcl2 dephosphorylation in REH cells, a role for PP2A is implicated. Recently, we have found that ceramide can activate the double-stranded RNA dependent protein kinase (PKR). Interestingly, PKR can phosphorylate the B56 a regulatory B subunit of PP2A resulting in enhanced PP2A activity. REH cells contain abundant levels of B56 a. Furthermore, B56 a co-localizes with Bc12 in mitochondrial membranes in REH cells. Thus ceramide activation of a Bc12 phosphatase may involve phosphorylation of B56 a by PKR. This finding raises the possibility that the B56 a regulatory subunit may contribute to the putative tumor suppressor properties of PP2A perhaps by a mechanism involving regulation of the potent tumor promoter, Bc12. In this proposal, we hypothesize a new model featuring the dynamic regulation of Bc12 function via ceramide-mediated activation of a Bc12 phosphatase by a mechanism involving PKR. To critically test this hypothesis we have identified two specific aims: (1) to determine the mechanism(s) by which ceramide activates and regulates the Bcl2 phosphatase and (2) to determine if the function of B56 alpha is regulated through a mechanism involving phosphorylation by PKR. State of the art molecular and biochemical methodologies will be employed including histochemical analysis, in vivo and in vitro expression studies, interactive cloning strategies to identify PP2A binding proteins, and the creation of a PP2A/B56 alpha transgenic mouse strain to genetically and functionally characterize PP2A in the context of the whole animal. The results are expected to fill in fundamental gaps in our knowledge regarding the signaling mechanism by which ceramide may regulate stress-signaling in cells expressing Bc12. From these results, it is expected that the development of novel anticancer strategies that target mechanisms regulating Bc12 function will occur.